Tuesday, June 23, 2015

6 23 2015 Flanking Primer PCR pt. 3

Today I ran a PCR on the two new flanking primers Actin and HSP70 as well as re run some primers that failed to sequence (HSPb11, PGEEP4, BMP2). After running PCR, I ran the products out on gels and then extracted the bands for sequencing.


1680Flk_HSP70_FWDGACCCGGATGTCCAGAGGAAJH6/9/20152060O.luridaHSP70 Flanking
1678Flk_Actin_FWDTCGAGGGAGGAAGAGGAAGCJH6/9/20152059O.luridaActin Flanking
1666Flk_HSPb11_FWDAGAATTGTCTGTGGAATCGAGCJH6/1/20152259O.luridaHSPb11 FlankingQ9Y547
1664Flk_PGE/EP4_FWDGCTCAACGAATTGCTCTACTCCJH6/1/20152259O.luridaPGE/EP4 FlankingP32240
1668Flk_BMP2_FWDGGCTGGCTGGATCGTCATJH6/1/20151860O.luridaBMP2 FlankingP12643

Reagent Table:
Reaction_ComponentsVolumeFinal Concentration
2x Apex Red12.5125
Forward Primer (10uM)0.55
Reverse Primer (10uM)0.55
Using the final concentration I mixed each master mix going from largest to smallest volume. 
I then pipetted 24 ul in each well followed by 1 ul of water or sample depending. 

Then I ran the following PCR program:

95 C5 min
95 C30 sec
55 C30 sec
72 C30 sec
repeat steps 2-4 40 times
72 C3 min
4 CHold
Once the PCR finished I ran 25 ul of each product on a 1.3% agarose gel

Gel Reagent Table
1X Low TAE175 ml
Agarose2.3 g
EtBr17.5 ul

  1. Add agarose to TAE.
  2. Microwave 1 minute stir
  3. Repeat until no particulate matter in solution
  4. Add EtBr while agarose still hot
  5. Gently pour in one corner of the gel cast until tray is full 
I then ran the gel at 100 v for 40 minutes. I placed it on the transilluminator to view any bands that may have formed. The gel is below

Gel Layout:

It appears the Actin flanking primer failed hard. The HSP70 primer seemed to do pretty well but still created several smaller bands. I carefully extracted only the largest band from the HSP70 samples to ensure that it was the right product. The rest of the primers worked like the had previously. 

Once the bands were cut out of the gel and placed in a Millipore Purification Column and centrifuged at 5,000 rcf for 10 minutes to collect the purified DNA. 

To send them off for sequencing I plated 4 replicates of 10 ul for each sample in the appropriate tube. On a second plate I plated two replicates of 3 uM primer working stock for each appropriate sample. These plates are now being held in the 4 C in 213 for sequencing. 

Sam's last sequencing came back today and it appears there are more primers that need to be re amplified and sequenced I will work on that tomorrow. I will also make sure to send off the TLR2.1 samples as they were overlooked in the last batch.  

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